In the field of clinical examination, analyses of various components in body fluids, such as blood, urine, saliva, cerebrospinal fluid, etc., offer guides to diagnosis of many diseases or objective judgement of the efficacy of a treatment.
A general method of these analyses is one called wet chemistry, in which a body fluid (specimen) and a reagent solution are put in a cuvette and stirred, the cuvette is incubated at 37.degree. C. for a given period of time, and a substance produced by the reaction of a specific component of the specimen is determined with an absorption photometer, a fluorophotometer, a turbidimeter, etc.
On the other hand, an analytical method called dry chemistry is being developed. This method is advantageous in that a reagent is supplied as a dry state, preparation of a reagent on analyses is not at all necessary, stirring is not necessary, no waste liquid occurs, and a very small amount of a specimen would be enough for analyses of many items. Dry chemistry has been made use of for instantaneous examinations in emergency laboratories of hospitals, nurses'offices in hospitals at night, or doctor's offices.
The dry analysis kit used in dry chemistry generally comprises a reagent layer and a supporting base plate. The reagent layer is prepared by infiltrating a reagent capable of reacting with a component in a specimen into a porous matrix such as paper, cloth, nonwoven cloth, meshes, membrane filters, sinters, and ceramics, followed by drying, or by applying a mixture of the reagent and a polymer binder kneaded with a solvent to a thin resin film, followed by drying. While the reagent layer cut into strips can be used as such, where an expensive reagent, such as an enzyme, a substrate, or a color former, is used, use of the strip as having a large effective area would be uneconomical, leading to a great increase in cost.
Accordingly, a reagent layer is cut into 4 to 10 mm squares or rectangles, taking into consideration the size allowing visual colorimetry, the diameter of a light beam used in reflective photometry, accuracy of measurement, and ease in handling in the preparation or on use. The cut reagent layer is fixed onto a base serving as base or grip with an adhesive, e.g., a double-sided adhesive tape, a paste adhesive or an instantaneous adhesive or a hot melt resin.
Moreover, of the dry analysis kits for dry chemistry, a so-called peel type test piece comprising a base having thereon a reagent layer and a releasable film layer having a sample measuring function in this order is used in some cases. Upon use, after a specimen is applied to the peel type test piece, the film layer is stripped off to observe the coloration of the reagent layer.
More specifically, a film layer capable of filtering out corpuscles of whole blood and measuring out an adequate amount of a specimen to the reagent layer is laminated on a reagent layer prepared by impregnating a matrix made of a thermoplastic resin or a non-thermoplastic substance with a reagent. When the dry analysis kit of this type is used, a specimen (whole blood) is applied on the film layer, the corpuscles and excess blood are wiped off the surface of the film layer, and the film layer is stripped to expose the reagent layer to observe the degree of coloration of the reagent layer. In some cases, the film layer is stripped without being wiped.
As stated above, the most commonly employed method for fixing a reagent layer directly to a base is fixation with a double-sided adhesive tape. However, double-sided adhesive tapes usually use polyacrylic resins, which contain no small amounts of polymerization initiators, monomers, stabilizers, plasticizers and wetting agents. On contact with the reagent layer, these components tend to react with the reagent in the reagent layer resulting in coloration or decomposition of the active ingredient. Therefore, strict selection of a double-sided adhesive tape has been required for each item.
Further, fixation with a double-sided adhesive tape has been accompanied with such disadvantages that the adhesive adheres to a processing machine to cause machine trouble and that the adhesive adheres to the surface of a reagent layer to make a part of the reagent layer unreactive with a specimen, resulting in unevenness of color formation.
In order to eliminate these problems associated with a double-sided adhesive tape, fixation with a hot-melt adhesive (an adhesive consisting of a thermoplastic resin which softens at 80 to 150.degree. C.) has been used. In this case, however, the whole reagent layer must be kept at 100 to 110.degree. C. for several seconds to melt the hot-melt adhesive, which entertains a fear of denaturation of the reagent, particularly proteins, such as an enzyme, an antibody and an antigen. Additionally, the hot-melt adhesives contain plasticizers, stabilizers, and the like similarly to the double-sided adhesive tapes, and these components have adverse influences on the reagent.
A method comprising enveloping a reagent layer in fabric or a web and fusion bonding both sides of the envelope with a hot-melt adhesive has been suggested as a solution to the outstanding problems, as disclosed in JP-B-53-6551 (the term "JP-B" as used herein means an "examined published Japanese patent application"). The method consisting of enveloping a reagent layer in a nylon mesh and bonding both sides thereof with a hot-melt adhesive succeeds in solving the above-described two problems. However, there is a fear of the nylon mesh's getting loose due to shocks during transportation and, as a result, the reagent layer tends to move or come off. In addition, the method is troublesome and costly.
JP-B-6-68488 discloses a method for preparing a composition for detection, which comprises interposing a thermoplastic resin layer between a reagent layer and a base and cutting the laminate by means of a laser beam or ultrasonic waves to fix the cut area through fusion. This technique is for preparing a multi-layer kit for dry analysis without using an adhesive. However, the kit prepared is of the type that it is held by a clamping rod serving as a grip or placed on a holder on use. Further, margins cut off by a laser beam or ultrasonic waves go to waste. Moreover, a machine generating a laser beam or ultrasonic waves of sufficient power for cutting the laminate is required, and such a machine is generally expensive.
In particular, where a reagent layer to be fixed on a base is glass fiber filter paper, etc. having chemically bonded thereto an antibody, an antigen, an antibody-avidin-biotin complex, etc., which is used for microanalysis utilizing immune reaction (so-called dry immunoassay), use of a double-sided adhesive tape or a hot-melt adhesive gives rise to not only the above-mentioned problems but another problem that an unreacted component or a substance having an influence on the reaction is non-specifically adsorbed on the glass fiber filter paper to cause a great error.
Further, since the peel type test piece comprises at least three layers, the preparation process is troublesome because of involvement of two steps; one for adhering the first layer to the second layer, and then one for adhering the third layer to the second layer.
Furthermore, on removal of the film layer, it is necessary to be peeled between the film layer and the reagent layer. It is inconvenienced that the test pieces suffered peeling at the interface between the base and the reagent layer. Taking into consideration on this point, the adhesive strength between the reagent layer and the base should be stronger than the adhesive strength (hereinafter referred to as "interlaminar strength") between the reagent layer and the film layer. To make a difference in interlaminar strenth between the two adhesive interfaces is troublesome.
The three layers laid one on the other can be adhered at a time while making a difference in interlaminar strength between the two adhesive interfaces by use of two kinds of adhesives. However, components used in adhesives tend to give adverse influences to the reagent in the reagent layer as mentioned above. Therefore, use of adhesives is not favorable.
Hence it has been demanded to develop an ideal method for preparing a peel type test piece, by which the three layers can be fixed at a time without using an adhesive while making a difference in interlaminar bond strength.